Project Summary The brain is responsible for processing multiple modalities of sensory information, controlling motor output, and mediating higher-order cognitive functions. Brain asymmetry is a conserved feature of vertebrate nervous system and is thought to be an advantageous mechanism for efficiently processing information. It displays anatomical and molecular left-right (L-R) asymmetries that correlate with their functional specialization in particular cognitive processes. Hemispheric specialization for a given cognitive function reflects differences in the neural circuits of specific neuron clusters. Although tremendous progresses have been achieved to decipher the complex neural networks using single gene lineage tracing or transcriptome analysis, the heterogeneity and the complexity of L-R asymmetrically distributed, functional neural networks are still largely unknown. Recently, by using an inducible CD133 lineage reporter mouse line, we found that E8.5-labeled, CD133+ neural stem cell (CD133+ NSC) lineages distributed asymmetrically in L-R neocortex of the brain, suggesting potential involvement of CD133+ NSCs in L-R asymmetrical brain development. In this proposal, by employing droplet-based single cell transcriptome analysis, in situ hybridization, immunostaining, brain- clearing assay, and lightsheet microscopy, we aim to identify the cellular and molecular signatures and spatiotemporal distributions of E8.5-labeled, CD133+ NSC lineages that contribute to L-R asymmetrical cortical development. This work will shed some light on using lineage specific stem cells combined with single cell transcriptome analysis to build a comprehensive road map to the understanding of L-R neural connections in the brain.